Fluorescence imaging is particularly powerful because the use of fluorescent labels can yield a high specificity and because appropriate analysis of the fluorescence signal can provide information about the environment of the fluorophore molecules as well as their location. Although fluorescence imaging is a long-established technique, recent advances in both label and detection technology are radically enhancing its impact in biomedicine. For cell biology, genomics, and proteomics, for example, the use of fluorescence labels allows biologists to observe the location of specific oligonucleotides in an assay or proteins in living cells. For medical imaging, fluorescence can provide greater specificity than absorption- or scattering-based techniques because it can derive functional contrast from intrinsic autofluorescence and/or from exogenous fluorescence labels. As the understanding of cell biology, immunology and proteomics grows, the techniques developed for these research agendas will increasingly be applied directly in clinical practice. In particular, we are now seeing rapid growth in techniques to realize in vivo microscopy and high-resolution endoscopy, optical imaging tools that will make use of fluorescence to provide functional contrast.
Fluorescence microscopy, in which the sample absorbs incident photons and emits light (fluorescence) at different (longer) wavelengths, is a well-established way of providing contrast that is not achievable with reflected light microscopy. Since the wavelength of the emitted fluorescence depends on the energy level structure of the fluorescent molecules (or “fluorophores”), it may be used to distinguish different molecular species in a sample. Thus, qualitative imaging of fluorescence intensity can reveal the location or distribution of fluorophores and spectrally resolved intensity imaging can contrast different fluorophores if their fluorescence emission spectra are sufficiently different. In biological samples, almost all the constituents will exhibit fluorescence if excited at an appropriate wavelength. For example, ultraviolet radiation can excite many organic molecules. This is often a problem since the aim is usually to study one or two specific biological entities and normally the excitation wavelength is carefully chosen so that it only excites the molecular species of interest. At longer wavelengths, in the visible and near infrared, it is not always possible to find an excitation wavelength to excite a given biological sample—particularly given the lack of tunable visible lasers suitable for microscopy. In many cases it is convenient to label the biological target with a fluorescent molecular species that can be excited using a convenient light source. Fluorophore labels are usually selected for their high fluorescence efficiencies and for their potential to be localized or attached to the biological targets in the sample. The latter issue is nontrivial and has engaged the attention of the biology and chemistry communities for many decades.
Quantitative imaging of fluorescence intensity can furnish functional information about a sample since the efficiency of the fluorescence process can reveal information about the fluorophore and its local environment. Fluorescence efficiency is conveniently parametenzed by the quantum efficiency η, defined as Γ/(Γ+k), where Γ and k are, respectively, the radiative and nonradiative decay rates. The quantum efficiency may also be described as the ratio of the number of fluorescence photons emitted to the number of excitation photons absorbed. The radiative decay rate is related to the transition oscillator strength, while the nonradiative decay rate can vary according to how the fluorophore interacts with its local environment. For some fluorophores, k is sensitive to the local pH, or to calcium ion concentration, or to physical factors such as viscosity. For this reason, fluorescent “probes” may be employed to produce functional maps of perturbations in such environmental factors by recording distributions of changes in k using quantitative fluorescence intensity imaging. In heterogeneous biological samples, however, it can be difficult to measure quantum efficiency because of possible artifacts arising from, for example, nonuniform fluorophore concentration, excitation flux or detection efficiency. Optical scattering can also degrade quantitative intensity measurements. For carefully prepared thin samples, one can assume that some of these factors are uniform across the field of view but for thick samples, and particularly for in vivo imaging, it is often not possible to reliably determine quantum efficiency. A more robust approach is to make relative intensity measurements at each pixel in the field of view. One approach is to employ special probes with fluorescence spectra that change in a predictable way according to the strength of the local environmental perturbation—the concentration of Ca+2 ions, for example. By incorporating such probes into a sample and recording fluorescence intensity images at two or more wavelengths, it may be, for example, possible to produce a map of Ca+2 concentration distributions in order to study signaling in nerve synapses. Such spectrally resolved relative measurements could effectively eliminate factors caused by optical scattering and noise from background fluorescence, as well as nonuniformity in fluorophore concentration, excitation and detection efficiency. “Wavelength-ratiometric imaging” has been successfully applied to microscopy and to in vivo diagnostic imaging. Unfortunately, this technique is limited to those instances for which suitable wavelength-ratiometric probes (or endogenous fluorophores) are available, currently a significant restriction.
Another way of obtaining functional information from relative intensity measurements (or images) is to temporally resolve fluorescence profiles after pulsed (or modulated) excitation. Just as spectral discrimination adds both a new dimension to fluorescence data and enhanced opportunities for contrast and robust functional imaging, the application of temporal resolution adds yet another dimension. Fluorescence lifetime imaging (FLIM) involves determining the average fluorescence decay time for each pixel in the field of view and producing a map (or series of maps) of lifetime data. Like the quantum efficiency, η, the fluorescence lifetime (τ) depends on both the radiative and nonradiative decay rates. Like η, it can be used to contrast different fluorophore species (via k) and different local fluorophore environments (via k). FLIM thus provides a robust functional imaging modality that may be applied to any fluorescent sample, and it is currently being applied to biological samples ranging from single cells to bulk tissue. FLIM is also attracting growing interest among researchers active in the field of microanalysis of sample arrays and high-throughput screening.
Water-stabilized nanocrystals capable of conjugating to probe molecules are particularly attractive for fluorescence based biological imaging and detection because they have a continuously selectable wavelength emission (i.e. many colors are realizable). Probe molecules have a specific affinity toward a target molecule and include proteins, avidin, streptavidin, biotin, nucleic acids, antibodies, enzymes, aptamers, oligonucleotides etc. The resultant nanocrystal tagged probe is used in a biological assay to optically identify the presence of a target molecule within the sample. Evident Technologies Inc. has recently demonstrated that water-stabilized nanocrystals are ideally suited for fluorescence lifetime imaging as well. All nanocrystal populations, despite their average size (which determines the emission wavelength) have nearly the same fluorescence lifetime of 15–20 nanoseconds (ns). This lifetime is very large when compared to the fluorescence lifetime of organic fluorophores and especially when compared the autofluorescence lifetime of organic molecules within a sample. Thus, using water-stabilized nanocrystal complexes coupled to a specific binding molecule, one can perform a biological assay or image using a time resolved detection platform that has an enhanced signal to noise ratio over that of conventional fluorescence detection methodologies.
Semiconductor nanocrystals are tiny crystals of II–VI, III–V, IV–VI materials that have a diameter typically between manometer (nm) and 20 nm. In the strong confinement limit, the physical diameter of the nanocrystal is smaller than the bulk exciton Bohr radius causing quantum confinement effects to predominate. In this regime the nanocrystal has both quantized density and energy of electronic states where the actual energy and energy differences between electronic states are a function of both the nanocrystal composition and physical size. Larger nanocrystals have more closely spaced energy states and smaller nanocrystals the reverse. Because interaction of light and matter is determined by the density and energy of electronic states, many of the optical and electric properties of nanocrystals can be tuned or altered simply by changing the nanocrystal geometry (i.e. physical size).
Single nanocrystals or monodisperse populations of nanocrystals exhibit unique optical properties that are size tunable. Both the onset of absorption and the fluorescence wavelength are a function of nanocrystal size and composition. The nanocrystals will absorb all wavelengths shorter than the absorption onset and emit light (at a wavelength corresponding to the absorption onset. The bandwidth of the fluorescence spectra is due to both homogeneous and inhomogeneous broadening mechanisms. Homogeneous mechanisms include temperature dependent Doppler broadening and broadening due to the Heisenburg uncertainty principle, while inhomogeneous broadening is due the size distribution of the nanocrystals. Populations of nanocrystal with a narrow size distribution have, as a result, a narrow FWHM of emission spectra. The quantum yield (i.e. the percent of absorbed photons that are reemitted as photons) is influenced largely by the surface quality of the nanocrystal. Photoexcited charge carriers will emit light upon direct recombination but will give up the excitation energy as heat if phonon or defect mediated recombination paths are prevalent. Because the nanocrystal has a large surface area to volume ratio, dislocations present on the surface or adsorbed surface molecules having a significant potential difference from the nanocrystal itself will tend to trap excited state carriers and prevent radiative recombination and thus reduce quantum yield. Quantum yield can be increased by removing surface defects and separating adsorbed surface molecules from the nanocrystal by adding a shell of a semiconductor with a wider bulk bandgap than that of the core semiconductor.
Hines and Guyot-Sionest developed a method for synthesizing a ZnS shell around a CdSe core nanocrystal. See Hines M., Guyot-Sionnest P., Synthesis and Characterization of Strongly Luminescent ZnS-Capped CdSe Nanocrystals, J. Phys. Chem., 1996, vol. 100, no. 2, pp. 468, incorporated by reference herein. The CdSe cores, having a monodisperse distribution between 2.7 nm and 3.0 nm (i.e. 5% size distribution with average nanocrystal diameter being 2.85 nm), were produced using the Katari and Alivisatos variation of the Murray synthesis. The photoluminescence spectra of the core show a FWHM of approximately 30 nm with a peak at approximately 540 nm. The core CdSe nanocrystals were separated, purified, and resuspended in a TOPO solvent. The solution was heated and injected with Zinc and Sulphur precursors (Dimethyl Zinc and (TMS)2S) to form a ZnS shell around the CdSe cores. The resultant shells were 0.6±0.3 nm thick, corresponding to 1–3 monolayers. The photoluminescence of the core-shell nanocrystals had a peak at 545 nm, FWHM of 40 nm, and a quantum yield of 50%.
One problem, however, is that semiconductor nanocrystals are inherently insoluble in any solvent and require a coating with suitable functional groups to enable suspension. Therefore, coatings having hydrophilic groups are required for water solubility. Those same hydrophilic groups also act as anchoring sites to couple the nanocrystal to a tertiary molecule such as a protein, antibody, nucleic acid, polymer etc. Short chain thiols such as 2-mercaptoethanol and 1-thioglyceral have been used as stabilizers in the preparation of water-soluble CdTe nanocrystals. See Rajh et al., Synthesis and Characteristics of Surface-Modified Colloidal CdTe Quantum Dots, J. Phys. Chem., vol. 97, No. 46, 11999–12003, 1993; Rogach et al, Synthesis and Characterization of Thiol-Stabilized CdTe Nanocrystals, Ber. Bunsenges. Phys. Chem, vol. 100, No. 11, 1772–1778, 1996, incorporated by reference herein. Bawendi et al., describes a method of preparing water soluble nanocrystals that do not demonstrate a reduction on quantum yield using long chain multidentate thiols, however the nanocrystals will precipitate when dialyzed indicating a lack of tight binding to the nanocrystal surface. See U.S. Pat. No. 6,319,426, entitled “Water-Soluble Fluorescent Semiconductor Nanocrystals,” incorporated by reference herein. It is important to note that in biological assays that require the nanocrystal to couple to a probe molecule, the lack of tight coupling between the probe molecule and the nanocrystal surface will inevitably lead to the probe molecule becoming disassociated resulting in inaccurate results of the assay.